1. Field of the Invention
This invention relates to insulin-secreting .beta. cells and methods of producing .beta. cell populations having desirable features.
2. Description of Art
The insulin producing tissue of the pancreas, the islets of Langerhans, constitute a small fraction of the organ. Islets are largely composed of small clusters of .beta.-cells and there is a need to develop a reliable source of .beta.-cells which respond to glucose stimulation in a manner similar to that of normal islet cells for diabetes research and for implantation into diabetic subjects.
Populations of .beta.-cells are known to show considerable heterogeneity in their morphology, their insulin secretion, and their glucose responsiveness. Normal islet tissue has been found to contain .beta. cells which secrete insulin in response to glucose as well as some that do not. Individual glucose-responsive cells in the population have been shown to secrete insulin at different glucose levels. Pipeleers, "Perspective in Diabetes Heterogeneity in Pancreatic .beta.-Cell Population", Diabetes (1992) 41:777-780. Islet cells in primary tissue culture show a characteristic sigmoidal curve upon glucose stimulation. As indicated in Wollheim, et al., "Establishment and Culture of Insulin-Secreting .beta. Cell Lines", Methods in Enzymlogy (1990) 192:223-235, in a native .beta. cell from all but the ruminants, the half-maximum level of insulin secretion is at about 7-8 mM glucose and the maximum is at a glucose level of about 15 mM or so (meaning that insulin secretion is about twice as much at 15 mM glucose as at 7-8 glucose). It is desirable, thus, that cells designed to mimic the activity of normal cells, either for implantation or for testing show a similar pattern of insulin secretion.
Historically, .beta.-cells have been obtained by isolating them from primary tissue, employing collagenase digestion of the pancreas, a time-consuming and expensive process. However, while primary hormone-secreting cells can often be maintained for several months in culture, they generally undergo few or no cycles of cell division. During this time, the cells generally display a decrease in hormone secretion and/or a loss of regulation. For human transplantation purposes, researchers have investigated the use of both human and animal tissue. A major problem with the use of human tissue, however, is the shortage of available organs. Where animal tissue is used, extreme care must be taken to obtain material from pathogen-free animals and all isolated tissue must be extensively tested. Wang, et al., "Glucose- and Acetylcholine-Induced Increase in Intracellular Free Ca.sup.2+ in Subpopulations of Individual Rat Pancreatic .beta.-Cells", Endocrinology (1992) 131:146-152 and Wang, et al., "Glucose-induced Insulin Secretion from Purified .beta.-Cells", The Journal of Biological Chemistry (1993) 268:7785-7791 and others have sorted .beta. cells from other pancreatic tissue by fluorescence-activated cell sorting using inherent light-scattering patterns and flavin adenine dinucleotide autofluorescence. De Krijger, et al., "Enrichment of Beta cells from the human fetal pancreas by fluorescence activated cell sorting with a new monoclonal antibody", Diabetologia (1992) 35:436-443 has sorted human islet cells from other human pancreatic tissue by producing mouse monoclonol antibodies specific to the islet cells. The antibody was labeled and used for fluorescence-activated cell sorting, the resulting cultures showing an enriched .beta. cell content. In general, though, these cells do not divide, and it is costly and time-consuming to repeatedly prepare .beta. cells in this manner.
Some reports have indicated that .beta. cells isolated from primary tissue can be made to divide in vitro. For example, Brothers, in "Hormone-Secreting Cells Maintained in Long-Term Culture", PCT Application WO 93/00441 published Jan. 7, 1993, selected and cultured cells from human pancreatic tissue without use of collagenase or centrifugation to establish subcultures of glucose-responsive cells cultured, at least originally, in media resembling the in vivo environment. Subsequently, individual cells or cell clumps in culture were selected for further propagation according to proliferation rate and amount of insulin secreted. Thawed cells which had been cryopreserved and cultured at passage 47 were tested for insulin secretion. Insulin secretion, according to the data presented, did not show the characteristic sigmoidal curve of correctly regulated cells, but rather what appears to be a horizontal line showing relatively static insulin secretion at different glucose levels. In addition, insulin levels are only at about 3.7 .times.10.sup.3 .mu.IU/1.5.times.10.sup.6 cells/hour. Furthermore, these cultures were not free from contaminating non-.beta. cells.
Zayas, et al., in "Proliferated Pancreatic Endocrine Cell Product and Process", EPO Application A2 0 363 125, published Apr. 11, 1990, discloses the culturing of pancreatic islet progenitor cells. These cells were proliferated in subculture in a collagen/laminate substrate gel to allow a three-dimensional culture system. The undifferentiated progenitor cells, when implanted, are reported to differentiate in vivo, resulting in in vivo insulin secretion.
Other researchers have attempted to overcome the problems associated with isolating natural islet cells by developing .beta.-cell lines. A cell line offers several advantages over the use of primary tissue, as it provides a renewable source of cells having consistent properties. Attempts have been made to develop reliable cell lines from insulinomas. Wollheim, et al., supra, reports that a major problem with such cell lines, though, is the tendency of these cells to lose their differentiated status in culture, and a corresponding decrease in the cellular insulin content. As a result, most such previous approaches have achieved only limited success. After repeated passaging in vitro, these cell lines tend to show little or no insulin secretion, and/or a lack of desired insulin regulation in response to glucose.
Gazdar, et al., "Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor", Proc. Natl. Acad. Sci. USA (1980) 77:3519-3523 discloses the establishment of cell lines of rat pancreatic islet cells devoid of fibroblastoid cells by centrifuging to remove erythrocytes and enhancing growth by using feeder layers of rat liver cells. The final cultures were well-isolated colonies harvested and propagated to mass cultures. However, different sublines of the cell lines show different amounts of glucose responsiveness, and maximum insulin production shown after about 100 days was about 150 to 250 .mu.U/10.sup.6 cells/24 hours.
.beta.-cell lines have been developed from X-ray induced mouse insulinomas as well as from insulinomas in transgenic mice expressing simian virus 40 T antigen. See Asfari, et al., "Establishment of 2-Mercaptoethanol-Dependent Differentiated Insulin-Secreting Cell Lines", Endocrinology (1992) 130:167-178, Hanahan, "Heritable formation of pancreatic .beta.-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes", Nature (1985) 315:115-122 and Efrat, et al., "Glucose Induces insulin Gene Transcription in a Murine Pancreatic .beta.-Cell Line", The Journal of Biological Chemistry (1991) 66:11141-11143. However, these cells (specifically RIN cells, HIT cells, .beta.-TC cells, and INS cells) either do not show high insulin secretion or correct regulation and frequently do not retain their secretory characteristics over numerous passages. A discussion of the .beta.-TC-3 cell line is found in detail in Efrat, et al., Supra and numerous .beta.-TC cell lines are discussed in D'Ambra, et al., "Regulation of Insulin Secretion from .beta.-Cell Lines Derived from Transgenic Mice Insulinomas Resembles that of Normal .beta.-Cells", Endocrinology (1990) 126:2815-2822. Although INS cells, particularly INS-1 show some degree of regulation, they do not show a large increase between half-maximum and maximum secretion and lower levels of secretion found in correctly regulated cells. In addition, the INS cells are mercaptoethanol-dependent for growth. Asfari, supra.
Some researchers have made specific attempts to overcome various of these problems. Miyazaki, et al., "Establishment of a Pancreatic .beta. Cell Line That Retains Glucose-Inducible Insulin Secretion:Special Reference to Expression of Glucose Transporter Isoforms", Endocrinology (1990) 127:126-132 discloses two .beta.-cell lines, called MIN6 and MIN7 obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice, the former obtained using "more than one cloning step", specific teachings of these steps being absent from the article. These cells have been characterized at 16-23 passages by Sakurada, et al., "Relation between Glucose-Stimulated Insulin Secretion and Intracellular Calcium Accumulation Studied with a Superifusion System of a Glucose-Responsive Pancreatic .beta.-Cell Line MIN6 ", Endocrinology (1993) 122:2659-2665 and Ishihara, et al, "Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets, Diabetologia (1993) 36:1139-1145. Additional information about these cells is found in Hamaguchi, et al., "NIT-1, a Pancreatic .beta.-Cell Line Established From a Transgenic NOD/Lt Mouse", The Jackson Laboratory, Bar Harbor, Me. (1991). According to Miyazaki, the MIN6 cells are regulated at 30 passages, although no data is presented to characterize the quality of regulation. It is interesting to note, as well, that while Ishihara has also characterized the MIN6 cells, and shown regulation at passages 16 to 23, the insulin output was significantly lower than initially reported by Miyazaki for these same cells at passage 16, suggesting some deterioration in insulin secretory response. However, at best these cells are reported to secrete about 1125 .mu.IU of insulin/45 min/10.sup.5 cells.
Increased intracellular free Ca.sup.+2 ("cytosolic free calcium") is known to be induced by glucose in certain .beta. cells. According to Wang, et al., "Glucose- and Acetylcholine-Induced increase in Intracellular Free Ca.sup.2+ in Subpopulations of Individual Rat Pancreatic .beta.-Cells", Endocrinology (1992) 131:146-152, p. 149, the pattern of response in .beta. cells is similar to that of whole islets and. isolated pancreas cells in prior studies. Wang, et al., "Glucose-induced Insulin Secretion from Purified .beta.-Cells", The Journal of Biological Chemistry (1993) 268:7785-7791 has shown that .beta. cells which do not show increased calcium concentration in direct response to glucose only may do so in the presence of other agents, resulting in increased insulin secretion in response to glucose stimulation. The presence of cytosolic free calcium in MIN6 cells (shown to be correctly regulated) and RINm5F cells (which have not shown high insulin secretion) was investigated by Sakurada, et al., supra. A close relationship between the rise of cytosolic free calcium concentration and insulin secretion was reported.
Omann, et al., "Pertussis Toxin Effects on Chemoattractant-Induced Response Heterogeneity in Human PMNs Utilizing Fluo-3 and Flow Cytometry", Cytometry (1991) 2:252-259 discloses the use of Fluo-3-acetoxymethyl ester (produced by Molecular Probes, Eugene, Ore.), hereafter sometimes referred to as "Fluo-3", which binds with Ca.sup.+2 in polymorphonuclear leukocytes for measurement of cytosolic calcium levels induced by N-formylpeptide.
Attempts have been made to transplant both insulinoma and normal islet cells into insulin-requiring organisms. The insulinoma transplanted into rats at an extrapancreatic site by O'Hare, et al., "Influence of a transplantable insulinoma on the pancreatic status of insulin and pancreatic polypeptide in the rat", Diabetologia (1985) 28:157-160 resulted in insulinaemia and hypoglycemia compared with controls. Undifferentiated pancreatic islet progenitor cells were transplanted into mice and allowed to differentiate in vivo for insulin production in vivo in Zayas, et al., supra.
The implantation of islet cells is discussed generally in Lacy, "Status of islet cell transplantation", 1 Diabetes Reviews (1993) No. 1, pp. 76-92. According to Lacy, to reduce rejection of foreign cells in the host organism, certain attempts have been made to reduce contact of the foreign cell with the host. For example,, fetal rat islet cells encapsulated in microspheres have been transplanted into mice. Biocompatibility problems encountered were reduced by coating the microspheres with alginate. According to Lacy, mouse pancreatic cells encapsulated in hollow fibers had prolonged survival when transplanted into hamsters. Lacy indicates that suspending rat islets in alginate, however, while encapsulated in acrylic copolymer hollow fibers has been shown to maintain normoglycemia in diabetic mice, using even a subcutaneous site, normally a deleterious one for islet cells. In addition, Hoffman, et al., in Experimental Neurology, "Transplantation of a Polymer-Encapsulated Cell Line Genetically Engineered to Release NGF", (1993) 122:100-106, reports that the transplantation of rat fibroblasts or fibroblasts genetically modified to produce NGF (nerve growth factor) were loaded within a thermoplastic hollow fiber-based capsule.
However, in Hicks, et al., "Transplantation of .beta. cells from transgenic mice into nude athymic diabetic rats restores glucose regulation", Diabetes Research and Clinical Practice (1991) 14:157-164, .beta.-cells from the mouse pancreatic .beta.-cell line .beta. TC-1, one of the cell lines mentioned above (which does not show proper regulation and shows low insulin secretion according to D'Ambra, Supra) attached to a collagen microcarrier and implanted in diabetic rats show improved insulin production and glucose response over diabetic rats implanted only with microcarriers, but showed increased granuloma formation and intense inflammatory reaction compared to diabetic controls without any implants.
It is thus apparent that there is still a need for the development of dividing .beta.-cell populations resembling normal islet cell populations in insulin secretion levels and in correct insulin regulation in response to glucose, particularly such cells which are phenotypically stable over time and which can be repeatably and predictably produced, as well as implanted for the treatment of diabetes.
The references discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or that they are otherwise part of the prior art.